Cardiovascular disease is the leading cause of death in the developed world. Thrombosis, or blockage of a blood vessel, is the cause of (1) myocardial infarction, ischemic stroke, mesenteric arterial disease, peripheral (limb) arterial disease; (2) venous thromboembolism (deep vein thrombosis and pulmonary embolism); (3) immune-mediated thrombocytopenia and thrombosis disorders (sepsis, heparin-induced thrombocytopenia, anti-phospholipid syndrome, thrombotic thrombocytopenic purpura, thrombotic complications of therapeutic monoclonal antibodies); and (4) miscellaneous common disorders classified with the acronym MCCATS (Malformation, Cancer, Cardiac, Artificial surface, Trauma, and Sickle cell disease). Each of these four categories individually accounts for around 500,000 deaths per year in the US alone, and countless others worldwide.
Thrombosis requires formation of a platelet-fibrin plug. In short, blood cell platelets combine with the plasma coagulation factors including fibrinogen and thrombin, which clot and ultimately lead to a blockage in the vessel. This vessel could be large—an artery or vein, or smaller—an arteriole, venule, or capillary. A thrombus cannot form without the platelets being activated in the body. A common activation mechanism for platelets is activation of a protein tyrosine kinase, Syk.
Activation of platelets occurs through activation of one or more receptors on the platelet. The major classes of receptors are G protein-coupled receptors, Immunotyrosine activation motif (ITAM) receptors, and adhesion receptors among which are integrins.
The presence of cardiovascular diseases, especially thrombosis-related diseases, has resulted in the development of drugs to mitigate and treat these diseases and conditions. Prior drugs and methods of treating thrombosis focus on preventing or treating platelet activation and has centered on the molecules that turn platelets on. Cyclooxygenase, ADP receptors, thrombin receptors and the integrin αIIbβ3 turn on platelet activation. FDA-approved medicines that target them are aspirin, clopidogrel, vorapaxar and abciximab, respectively. However, as is readily known to those of skill in the art, these drugs have serious concerns in some patient populations wherein these drugs are ineffective in many patients or they cause serious bleeding. Recently, Syk has been validated as a new target to mitigate platelet activation and thrombosis4,61.
Based on the four classes of thrombosis, the immune-mediated thrombocytopenia and thrombosis disorders present a specific opportunity as such patients are usually already in the care of physicians. Thus, there is a particular opportunity to identify and treat these patients at an early stage to reduce or eliminate morbidity. In certain cases, between about 0.1 to 3% of patients exposed to a form of the drug heparin develop heparin-induced thrombocytopenia (HIT). Patients developing HIT are at a significant risk of low platelet count and thrombosis. About 30 to 70% of untreated HIT patients develop venous or arterial thrombi that are life and/or limb threatening, which are caused by the formation of IgG antibodies against the heparin-PF4 complex. This complex activates platelets via FcγRIIA receptors, which results in thrombocytopenia and thrombosis.
Multiple Fcγ receptors for IgG antibody are present in humans. Among them, FcγRIIA, encoded by the FCGR2A gene, is the only one present on human platelets.5 Prior studies demonstrated that platelet FcγRIIA was necessary for Heparin-induced thrombocytopenia (HIT) development in vivo with our human FcγRIIA/PF4 transgenic mouse model.5 Binding of the Fc portion of IgG in immune complexes or cross-linking FcγRIIA promotes phosphorylation of tyrosine residues in the immunoreceptor tyrosine-base activation motifs (ITAMs), which further provides binding sites for the Src homology 2 (SH2) domains in Syk. Multiple tyrosine phosphorylation events on Syk occur after FcγRIJA ITAM phosphorylation and Syk becomes an activated protein kinase. The signaling is further transmitted by phosphorylation of phospholipase Cγ2 (PLCγ2), phosphatidylinositide 3-kinases (PI3Ks), and the Linker for Activation of T cells (LAT), followed by calcium mobilization and protein kinase C activation. These signals ultimately lead to platelet activation and thrombus formation.7 Recently, FcγRIIA was also identified as a key regulator in platelet integrin outside-in signaling.6,8,9 Syk is naturally regulated by platelet T-cell ubiquitin ligand 2 (TULA2). Increased TULA-2 levels will decrease Syk activity and platelet activation in thrombosis.